Methods for screening for a compound useful in the treatment or prevention of lymphocytic disorders, for inhibiting lymphocite activity and preventing or treating lymphocytic disorders

ABSTRACT

A method of screening for a compound useful in the treatment of a disease or condition characterized by an immune cells disorder, wherein said cell expresses NTPDases, said method comprising the steps of contacting a candidate compound with ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), wherein the candidate compound is selected if the activity of said NTPDase is reduced in the presence of the candidate compound as compared to that in the absence thereof. A method for inhibiting an immune cell activity in a mammal, comprising targeting immune cells with an effective amount of a NTPDase inhibitor. A method to prevent or reduce the risk of rejection of transplanted tissue or organ, comprising administering to said animal an effective amount of NTPDase inhibitor.

FIELD OF THE INVENTION

The present invention relates to methods for screening for a compounduseful in the treatment or prevention of lymphocytic disorders, forinhibiting lymphocyte activity and preventing or treating lymphocyticdisorders. More specifically, the present invention is concerned withNTPDase inhibitors and methods of using same.

BACKGROUND OF THE INVENTION

Many conditions and disease involve defective or undesirable Tlymphocyte proliferation or activation; these disease and conditionsinclude graft-host reaction such as organ or tissue rejection,neoplasia, tumors, T cell leukemia, immune diseases and various diseasesand conditions in oncology and oncohematology, etc.

In particular, the T cell leukemia is a T lymphocyte disorder: aprogressive or aggressive disease. The T cell malignancies can beclassified into two principal groups: 1-T cell acute lymphoblasticleukemia (ATLL) and 2-T cell chronic lymphoblastic leukemia.

Actual clinical therapies include chemotherapy, radiotherapy, bonemarrow transplantation, or a combination of this treatment. Patients arealmost always resistant to chemothereupic agent and were refractory toother therapies. ATLL is a very aggressive T cell malignancy for whichno successful treatment is yet available. The acute forms have a pooroutcome with overall survival ranging from 5 to 13 months. ATLL does notrespond or only transiently to combination chemotherapy.

Patients with chronic forms become refractory to therapy. The poorresponse to therapy related to chemotherapy resistance. Results are notsuperior with immunotherapy.

Adenosine triphosphate (ATP) and Adenosine diphosphate (ADP) act assignaling molecules for cells of virtually all origins. Indeed thesepurines, which are released from the cells by exocytotic andnon-exocytotic mechanisms can interact with specific receptors andthereby can influence the different physiological systems. One finds onthe cell surface two classes of receptors which respond to ATP and ADPand identified as purinoceptors, and one additional class ofpurinoceptors which respond to adenosine. In the immune system, the roleof purines is still poorly understood. It is well known however thatextracellular ATP can modulate certain responses of various lymphocytecell polulations such as DNA synthesis, blastogenesis and mediated cellkilling via specific cell membrane purinoceptors. In these respects, ithas been shown that ATP inhibits natural killer cell activity (11) ofhuman and murine origins and phagocytosis in mouse macrophages (3).Extracellular ATP can stimulate in vivo DNA synthesis of thymocytes butinhibits DNA synthesis of spleen and peripheral blood lymphocytes (6).Baricordi et al (7), reported that AT had a synergistic effect on DNAsynthesis stimulated by selective T-cell mitogens such as PHA oranti-CD₃ monoclonal antibody.

At least two functionally distinct purinoceptors subtypes have beendescribed in lymphotytes: 1) that coupled to G proteins, namely the P2Yreceptor linked to IP3 generation and Ca2+ mobilization fromintracellular stores and 2) the P2X/P2Z receptor which gates a channelpermeable to Na+ and Ca2+ (7). A P2Y receptor has been described inhuman B lymphocytes and has been reported to be absent from T cells(13-17). Whereas nucleotide activated ion channel has been shown to beexpressed to a low level in normal B-lymphocytes and to be upregulatedin resting mouse T and B-lymphocytes, leukemic peripheral bloodlymphocytes. According to Baricordi et al. (7), human peripheral bloodlymphocytes and purified T lymphocytes express a P2×7 purinoceptor andionic channel gated by extracellular ATP that is involved in the controlof mitogenic stimulation by different stimuli. The extracellularconcentration of the agonist (ATP, ADP or adenosine) which elicits thecellular response is determined by several parameters: 1) rate ofrelease and diffusion, ii) metabolism by ectonucleotidases and iii)binding affinity of the receptors. In this context, the role played byectonucleotidases, and more specifically ectonucleoside triphosphatediphosphorylase (NTPDase), appears to be determinant. In this respect,immuno competent cells, such as lymphocytes and macrophages, expressectonucleotidases activities. Ectonucleotidase which catalyses thehydrolysis of ATP to ADP has been reported to B cells (18), macrophages(20), natural killer (NK) cells and CTL (21). It has been proposed thatan ecto ATPase could protect murine CTL cells from the lytic effects ofextracellular ATP released during granules exocytosis (1, 10, 22) andthat this ecto ATPase was required for the cytolytic activity of NKcells. Recent data on ATPDase show that NTPDase 1 (CD₃₉), which isdistributed on the cell surface of many cell types, plays a key role inthe conversion of extracellular ATP to ADP and ADP to AMP. This enzyme,put in evidence many years ago by Lebel et al. (36) in pig pancreas, hasbeen recently identified in primary and secondary lymphoid organsincluding spleen, thymus, tonsils, and Peyer's patches and isolatedlymphocytes and macrophages from pig spleen (Benrezzak et al. (1999)).It has also been demonstrated that CD₃₉, a lymphoid cell activationantigen (30), corresponds to human NTPDase (31). Kansas et al. (32)cloned the latter molecule and studied its distribution and theyreported that this protein is expressed on activated NK, B, and T cellsof peripheral blood and is found in certain lymphoid tissues, namelytonsils and thymus. Despite these reports demonstrating the presence ofNTPDase (CD₃₉) in the immune system, the physiological role played bythe NTPDase in the immune response remains to be clarified. A relevantobstacle to the understanding of the NTPDase functions is a lack ofspecific inhibitors, i.e. an inhibitor that does not interfere withpurinoceptors. A recently described NTPDase inhibitor, BGO 136, anaphtyl derivative also known as 1-hydroxynaphtalene-3,6 disulfonicacid, produced a mixed type of inhibition with Ki_(s) of 380 uM with ATPand ADP as substrate, respectively (Gendron et al. 2001). Biochemicaland pharmacological characterisations of a further NTPDase inhibitor,the 8 Bus ATP, are described in Gendron et al. (2000), J Med Chem. TheU.S. patent application Ser. No. 09/591,177 in the name Beaudoin et al.teaches that various others compounds are also able to inhibit NTPDaseactivity.

There remains a need to determine the activity and functions of theNTPDase. In particular, there remains a need to determine whetherNTPDase has a direct implication in immune responses.

There also remains a need to provide new means of preventing or treatingdiseases or conditions involving defective or undesirable T lymphocyteproliferation or activation.

SUMMARY OF THE INVENTION

The present invention teaches that NTPDase is directly involved in theimmune response. It teaches that NTPDase is an agonist of T lymphocyteproliferation and humoral response. It also teaches that NTPDaseinhibitors are able to inhibit T lymphocyte proliferation and humoralresponse. More specifically, it teaches that NTPDase inhibitors may beused to prevent graft rejection.

More specifically, the present invention also teaches the inhibitoryeffects of BGO 136, erythrosin B, 8 Bus-ATP, 8 Bus-AMP on cell specificmitogenic stimulation and lymphoproliferative properties of T cells andon primary antibody response to a T-cell dependent antigen. Thesefindings emphasize the importance of NTPDase for the T-helper cell forfunctions in humoral responses induced by T cell dependent antigens.

Results obtained on mouse revealed efficacy of NTPDase inhibitors andtolerance for long-term treatment.

The present invention provides uses and methods of using NTPDaseinhibitors in the prevention or treatment of disease or conditioncharacterized by a T lymphocytic disorder including graft vs hostreaction following organ or tissue transplant, blood disorders such asneoplasia (leukemia and lymphoma), autoimmune disorders such asrheumatoid arthritis and psoriasis, and various other oncological andoncohematological diseases and conditions.

Composition within the scope of the present invention should contain theNTPDase inhibitor in an amount effective to achieve the desiredtherapeutic effect while avoiding adverse side effects. The dosage willbe adapted by the clinician in accordance with conventional factors suchas the extent of the disease and different parameters from the patient.Typically, 0.001 to 50 mg/kg/day of the NTPDase inhibitor in accordancewith the present invention will be administered to the mammal.Pharmaceutically acceptable preparations and salts of the NTPDaseinhibitor are within the scope of the present invention and are wellknown in the art (Remington's Pharmaceutical Science, 16th Ed., MackEd.).

Assays to Identify NTPDase of the Present Invention

Preferred methods for testing the ability of candidate compounds toinhibit the NTPDase activity are presented herein. It will be understoodthat the invention is not so limited. Indeed, often assays well known inthe art can be used in order to identify non-competitive, extracellularantagonists of the present invention.

It shall be understood that the “in vivo” experimental model can also beused to carry out an “in vitro” assay.

In Vitro Assays

In a preferred embodiment, candidate inhibitors are tested for theirability to inhibit NTPDase ability to modulate lymphocytic proliferationwith the incorporated triated thymidine method. Other ways of determinecellular proliferation may be use. For instance cell count and theassays described in Baker F. L. et al. (1995) Cell Prolif. 28(1): 1-15;Cheviron N. et al. (1996) Cell Prolif. 29(8): 437-46; Hu Z. W. et al.(1999) J: Pharmacol. Exp. Ther. 290(1): 28-37; and Elliott K. et al.(1999) Oncogene 18(24): 3564-73.

In Vivo Assays

The assays described above may be used as initial or primary screens todetect promising lead compounds for further development. Lead inhibitorswill be further assessed in additional, different screens. Therefore,this invention also includes secondary screens which may involve variousassays utilizing mammalian cell lines expressing these purinoceptors orother assays.

In yet other preferred embodiments, candidate inhibitors are tested fortheir ability to inhibit NTPDase's ability to elicit humoral responseand specifically antibody production. Any methods known in the artincluding those described herein for determining antibody production maybe used including the techniques involving the formation of hemolyticplaques, etc.

Tertiary screens may involve the study of the identified inhibitors inanimal models for clinical symptoms. Accordingly, it is within the scopeof this invention to further use an inhibitor identified as describedherein in an appropriate animal model such as a rat or a mouse. Forexample, a inhibitor can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an inhibitor identified as described herein can be usedin an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatment (e.g.treatments of different types of disorders associated with aderegulated, defective, undesirable immune cells activity), as describedherein.

More specifically, in accordance with the present invention, there isprovided a method of screening for a compound useful in the treatment ofa disease or condition characterized by an immune cells disorder,wherein said cell expresses NTPDases, said method comprising the stepsof contacting a candidate compound with ecto-nucleoside triphosphatediphosphohydrolase (NTPDase), wherein the candidate compound is selectedif the activity of said NTPDase is reduced in the presence of thecandidate compound as compared to that in the absence thereof. In aspecific embodiment, the contacting of the candidate compound with theNTPDase is performed in an immune cell selected from the groupconsisting in normal T lymphocyte, normal B lymphocyte, normal NK cell,normal macrophage, normal monocyte, Jurkat cell, Raji cell, Ramos cell,MonoMac™ cell, K562 cell and U937 cell.

According to a further aspect of the present invention, there isprovided a method for inhibiting an immune cell activity in a mammal,comprising targeting immune cells with an effective amount of a NTPDaseinhibitor. In specific embodiments, the method is further characterizedby one or more of the following characteristics: the immune cells arenormal lymphocytes (such as T and B lymphocytes) or tumoral immune cells(such as T or B neoplastic lymphocytes, and more specifically Jurkatcells), the activity is the T lymphocyte proliferation or the productionof antibodies. In more specific embodiments, the activity is inducedeither by organ or tissue transplant, by an allergen, or by autoimmunediseases. In further specific embodiments, the NTPDase inhibitor isselected from the group consisting of BGO 136, erythrosin B, nucleotideor nucleotide derivative including AMP, 8 Bus-AMP and 8 Bus-ATP, andanalogues thereof.

In a further aspect of the present invention, there is provided a methodto prevent or reduce the risk of rejection of transplanted tissue ororgan, comprising administering to the mammal an effective amount ofNTPDase inhibitor. In a specific embodiment, the NTPDase inhibitor isselected from the group consisting of BGO 136, erythrosin B, nucleotideor nucleotide derivative including AMP, 8 Bus-AMP and 8 Bus-ATP, andanalogues thereof.

In a further aspect of the present invention, there is provided acomposition for use as an immunosuppressive agent in graft transplantcomprising an effective amount of BGO 136 or BGO 136 analogue in apharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a use ofa BGO 136 or BGO 136 analogue in the making of a medicament for use asan immunosuppressive agent in graft transplant.

NTPDase is a family of enzymes which catalyse the sequential hydrolysisof the gamma and betaphosphate bonds of nucleoside tri and diphosphatesand characterized by homologous sequences (apyrase conserved regions).As used herein, the term “NTPDase” refers to any member of this family.

As used herein, the term “disease or condition characterized by animmune cell disorder” is meant to include disease and conditionscharacterized by defective or undesirable immune cell activity. Itincludes normal T lymphocytes proliferation and normal B lymphocyteshumoral response, any dysfunction in normal immune cells includingmonocytes, NK cells, and lymphocytes, neoplastic immune cellsproliferation, etc. Without limiting the generality of the foregoing,such diseases and conditions include graft vs. host reaction followingorgan or tissue transplant (including graft rejection), blood disorderssuch as neoplasia (leukemia and lymphoma), autoimmune disordersincluding rheumatoid arthritis, psoriasis, Crohn disease and certaindiabetes, oncological and oncohematological diseases and conditions.

As used, the terms “neoplastic”, and “tumoral” are used interchangeably.

As used herein, the term “NTPDase activity” is meant to include theenzyme's ability to hydrolyse ATP and ADP and any downstream effect ofthis enzymatic reaction including lymphocyte proliferation and humoralresponse.

As used herein, the term “lymphocytes” is meant to include normallymphocytes and neoplastic/tumoral lymphocytes. Without limiting thegenerality of the foregoing, this term is meant to include normal T andB lymphocytes such as those found in normal mammals, andneoplastic/tumoral T and B lymphocytes such as Jurkat, Rami and Ramoscells.

As used herein, the term “effective amount” is meant to refer to anamount of NTPDase inhibitor administered in a single dose or in multipledoses sufficient to reduce or abrogate the undesirable lymphocyteactivity in the cell or the animal to which it is administered whileavoiding adverse side effects. Without limiting the generality of theforegoing, the results presented herein demonstrate that an amount of4,3 mg/g of base weight of transplanted spleen is sufficient to reducethe spleen increase by 56%. It refers to the amount necessary to producea benefit to the cell or animal to which it is administered asdetermined by a person of ordinary skill in the art.

As used herein, the term “BGO 136 analogue” is meant to refer to anynaphtyl derivative having the ability to inhibit an NTPDase activity.Furthermore, the F. B. Gendron doctorate thesis submitted to theUniversity of Sherbrooke on inhibitors defines the conformationalcharacteristics that enable an analogue to possess inhibitory abilities.

As used herein, the term “AMP analogue” is meant to refer to anyadenosine 5′-monophosphate derivative having the ability to inhibit anNTPDase activity including 8-BUS AMP. Since AMP is the last member ofthe reaction, it is submitted that it is reasonably predictable that anyAMP analogue will inhibit NTPdase.

As used herein, the term “ATP analogue” is meant to refer to anyadenosine 5′-triphosphate derivative having the ability to inhibit anNTPDase activity including 8-BUS ATP. Gendron et al (2000a) and Nahum etal (2002) describe such ATP analogues.

As used herein, the term “erythrosin B” is meant to refer to any memberof the erythrosin family or erythrosin derivative having the ability toinhibit an NTPDase activity.

As used herein, the term “nucleotide derivative” is meant to refer toany nucleotide having been subjected to any modification preserving itsability to inhibit an NTPDase activity. Assays as described in Picher etal (1996) showed that modifications on the ATP phosphate chain preventATP hydrolysis consequently granting resulting molecules with inhibitoryactivities.

As used herein, the term “humoral response” is meant to include antibodyproduction.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows the presence (the 78 KDa and 56 KDa lines) of NTPDase inhuman lymphoid cells. In this figure, PBL: normal peripheral bloodcells; Ramos: B leukemia cells; Jurkat: T leukemia cells; K562:erytroleukemia cells; Raji: B leukemia cells; NK: natural killer cells;Monomac™: monocytes leukemia cells;

FIG. 2 shows the effect of BGO 136 on NTPDases activity of humanperipheral blood lymphocytes (PBL). Panels A and B show BGO 136's effecton the ATPDase and ADPDase functions, respectively. Eight (8) assayswere performed each in triplicate. NTPDase 1 and 2 (in a lesser extent)were used;

FIG. 3 shows the inhibitory effect of BGO 136 on human peripheral bloodlymphocytes (PBL) proliferation. Panel A presents results with a control(unstimulated cells); Panel B presents results with cells stimulated byConA™ and Panel C presents results with cells stimulated with LPS™.Three (3) experiments were performed each in quadruplicate. [inhibitor]:indicates inhibitor concentration;

FIG. 4 shows the correlation between NTPDase activity of cells subjectedto BGO 136 as presented in FIG. 2 and proliferation assay of peripheralblood lymphocytes (PBL) subjected to BGO 136 as presented in FIG. 3.Each set of point corresponds to a single concentration of the inhibitor(concentration used are 0, 1, 5 and 10 mM of BGO136);

FIG. 5 shows the effect of BGO136 on humoral response (antibodiesproduction) 21 days after a first injection (Panel A), 32 days afterfirst injection (Panel B); and 40 days after a first injection (PanelC). In this figure, DM: indicates that multiple doses of BGO136 weregiven; +Ag: mice received BSA as Ag T cell dependent; −Ag: mice receivedonly vehicle; BGO136 was given in a single dose at the mentionedconcentration (400 or 800 mM). Each group contained ten (10) mice;

FIG. 6 shows the effect of erythrosin B on NTPDase activity of humanperipheral blood lymphocytes (PBL). Six (6) experiments were performedeach in triplicate;

FIG. 7 shows the effect of erythrosin B on human peripheral bloodlymphocytes (PBL) proliferation. Three (3) experiments were performedeach in sextuplicate;

FIG. 8 shows the correlation between NTPDase activity of cells subjectedto erythrosin B as presented in FIG. 6 and proliferation assay ofperipheral blood lymphocytes (PBL) subjected to erythrosin B aspresented in FIG. 7. Each set of point corresponds to a singleconcentration of the inhibitor. Concentrations used are 0, 10, 30, 50and 100 uM of erythrosin B;

FIG. 9 shows the effect of erythrosin B on humoral response (antibodyproduction) 21 days after first injection (Panel A), 32 days after firstinjection (Panel B) and 40 days after first injection (Panel C). In thisfigure, DM Multiple doses of Erythrosin B were given; Groupe Ag: micereceived only BSA as Ag T cell dependant without inhibitor; Erythrosin Bwas given in a single dose as mentioned 0.25 g/kg or 0.5 g/kg. Eachgroup contain 10 mice;

FIG. 10 shows the inhibitory effect of 8-BuS ATP on NTPDase activity ofhuman peripheral blood lymphocyte. One (1) experiment was performed induplicate;

FIG. 11 shows the inhibitory effect of 8-BuS-ATP on the human peripheralblood lymphocytes (PBL) proliferation. Three (3) experiments wereperformed each in sextuplicate;

FIG. 12 shows the correlation between NTPDase activity of cellssubjected to 8-BuS-ATP as presented in FIG. 10 and proliferation assayof peripheral blood lymphocytes (PBL) subjected to 8-BuS-ATP aspresented in FIG. 11. Each set of point corresponds to a singleconcentration of the inhibitor. Concentrations used are 0, 10, 50 and100 uM of 8-BuS-ATP;

FIG. 13 shows the inhibitory effect of 8-BuS AMP on NTPDase activity ofhuman peripheral blood lymphocyte. Two (2) experiments were performedeach in triplicate;

FIG. 14 shows the inhibitory effect of 8-BuS-AMP on the human peripheralblood lymphocytes (PBL) proliferation. Three (3) experiments wereperformed each in sextuplicate;

FIG. 15 shows the correlation between NTPDase activity of cellssubjected to 8-BuS-AMP as presented in FIG. 13 and proliferation assayof peripheral blood lymphocytes (PBL) subjected to 8-BuS-AMP aspresented in FIG. 14. Each set of point corresponds to a singleconcentration of the inhibitor. Concentrations used are 0, 10, 30 and100 uM of 8-BuS-AMP;

FIG. 16 shows the inhibitory effect of beta-gamma methylene ATP onNTPDase activity of human peripheral blood lymphocytes (PBL). Two (2)experiments were performed each in triplicate;

FIG. 17 shows the inhibitory effect of beta-gamma methylene ATP on thehuman peripheral blood lymphocytes (PBL) proliferation. Three (3)experiments were performed each in sextuplicate;

FIG. 18 shows the correlation between NTPDase activity of cellssubjected to beta-gamma methylene ATP as presented in FIG. 16 andproliferation assay of peripheral blood lymphocytes (PBL) subjected tobeta-gamma methylene ATP as presented in FIG. 17. Each set of pointcorresponds to a single concentration of the inhibitor. Concentrationsused are 0, 50, 100 and 250 uM of beta-gamma Me-ATP; and

FIG. 19 shows the inhibitory effect of NTPDase inhibition on neoplasticT cell growth Results obtained with erythrosin B, BGO 136, AMP,beta-gamma methylene ATP, 8-BuS-ATP are shown in Panels A to E,respectively. For each inhibitor, two (2) experiments were performedeach in quadruplicate.

DESCRIPTION OF SPECIFIC EMBODIMENTS Isolation of Human Lymphocytes

Human lymphocytes were obtained from peripheral venous blood of normaland healthy medication-free volunteers. Fresh blood collected in EDTAglass tubes was layered into Histopaque™-1077 (a solution of Ficoll™ andsodium diatrizoate adjusted to a density of 1.077, Sigma, USA) andcentrifuged at 400 g for exactly 30 min, at room temperature. Duringcentrifugation, erythrocytes and granulocytes were aggregated by Ficoll™and rapidly sedimented, whereas lymphocytes remained at the plasmainterface. Plasma (the upper layer) was carefully removed to preventdisturbance of the buffy coat. The latter fraction rich in lymphocyteswas recovered with a pasteur pipette and washed twice with RPMI™ 1640medium by centrifugation, for 10 min, at 250×g. The final pellet wassuspended in fresh RPMI™ 1640 medium supplemented with 2 mM L-glutamine,10% foetal bovine serum inactivated at 56° C. for 30 min, andantibiotics: penicillin 100 units/nl, streptomucin 100 ug/ml andamphotericin 2.5 ug/ml. Cells were counted with the hemacytometer.Viability, tested with Trypan™ blue exclusion assay, was superior to90%. The cell preparation named PBL (for peripheral blood lymphocyte)was immediately used for NTPDase assays and for mitogenic responses tostimulators.

Maintenance of Lymphoblastoid Human Cell Lines

Human cell lines were given by Dr. Jana Stankova (Department ofImmunology, Université de Sherbrooke). Jurkat cells were derived fromhuman T cell leukemia. Raji and Ramos cells are B-cell lymphomas derivedfrom peripheral blood of patients with Burkitt lymphoma. MonoMac™-1 is acell line derived from the peripheral blood of a 64 year-old male withactive monocytic leukemia. K562, which is used as highly sensitivetarget for NK cell activity is an erythroleukemia cell line. U937 is apromonocytic myeloid cell and finally the Y2 T2 C2 (NY) cell is a humannatural killer cell line. These lymphoblastoid cell lines which grow insuspension as single cells without attachment to glass, were maintainedby passage in complete RPMI 1640 medium with 2 mM L-glutamine containing10-20% heat inactivated foetal bovine serum [V/V] and antibiotics (100units/ml of penicillin), streptomycin 100 ug/ml and 2.5 ug/ml ofamphotericin) at 37° C. in a humidified 5% CO₂/95% air atmosphere.Mono-Mac™ 1 cells were maintained in the same complete RPMI™ 1640 mediumsupplemented with 1× non-essential amino acids and 1 mM Na-pyruvate at37° C. with 5% CO₂.

Mitogen Stimulation and Proliferation Assay

A thymidine uptake assay was used to evaluate the effect of NDPaseinhibitors on the proliferative activity of normal lymphocytes.

Cell suspensions of fresh human peripheral blood lymphocytes (PBL) wereresuspended in RPMI™ 1640 medium supplemented with 10% foetal bovineserum at a concentration of 2×106/ml. Aliquots of 100 ml were plated ona Falcon™ flask (96-well) and stimulated with 10 ug/ml of (Con-A) or 40ug/ml of (LPS™) at a final volume of 0.2 ml/well. ConA™ and LPS™ wereused to stimulate preferentially T cells and B cells, respectively. Cellcultures±inhibitors were incubated for 48 h at 37° C. in a 5% CO₂atmosphere [³H] thymidine (1 ug/well, specific activity 5.0 ci/mmol) wasadded to each microplate in a volume of 50 ul. After an additional a 4 hincubation at 37° C., cells were collected with a cell harvester and ³Hthymidine incorporation was measured in triplicate samples by liquidscintillation counting.

NTPDase Assays

Prior to the assays, human normal lymphocytes as well as leukemic celllines, were washed three times with phosphate-free saline in 95 mM NaCl,1 mM CaCl₂, 2 mM MgCl₂ and 45 mM Tris —HCl buffer (pH 7.4). Enzymeassays were carried out at 37° C. in 1 ml of the following assay medium:95 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl₂, 5 mM glucose, 0.05% BSA, 5mM tetramisole and 45 mM Tris-HCl (pH 7.5). The reaction was started byadding the substrate (200 uM of ATP or ADP) and stopped with 250 ul ofthe Malachite green reagent. Controls were run with the enzyme addedafter the Malachite green reagent. Where indicated, 10 mM sodium azide(NaN₃) or 1 mM, 5 mM and 10 mM of BGO 136 were added directly to theassay mixture. Inorganic phosphate was estimated according to Baykov etal.⁽¹⁾ and enzyme activity was either expressed as nmoles of Pireleased/min/mg of protein which corresponds to mUnits⁽²⁾ or as nmolesPi/min/10⁶ cells. Where indicated, lymphoid cells were lysed by threefreeze thaw cycles in saline buffer and protein was measured with themethod of Bradford using bovine serum albumin as a standard (3).

Electrophoresis and Western Blotting

Lymphocytes from human peripheral blood (PBL) and lymphoblastoid celllines obtained as described above were lysed and protein was measured.Samples of 20 ug protein of whole cell lysate were applied to each wellof polyacrylamide gel. Electrophoresis was carried out under denaturingconditions (SDS-PAGE) in a 10% polyacrylamide gel. Protein wassubsequently transferred to immobilon-P sheets and immunoblotted. Arabbit antiserum (Kally) which recognizes isoform 11 of ATPDase was usedas primary antibody at a dilution of 1:10 000. The secondary antibody, amouse monoclonal anti rabbit IgG conjugated to alkaline phosphatase(1:10 000) was detected by chemiluminescence using the Immun-Star™substrate, according to the recommendations of the supplier(Bio-Rad/laboratories).

In Vivo Antibody Production Assay

Animal Treatments and Experimental Design

Immunity mice were primed with antigen by an intraperitoneal injectionof BSA as a T-dependent antigen. Briefly, BSA was resuspended in PBS (pH7.2) and emulsified in the same volume of complete Freund's adjuvant(CFA). 30 ug BSA in 100 ml PBS was given to each mouse. Four groups often mice were immunized.

-   Group 1: mice were injected i.p. with BSA alone with emulsified in    (CFA) Group: Ag.-   Group 2: mice were injected i.p. with BSA plus BGO 136 (400 mM) in    100 ml PBS, emulsified in (CFA) or erythrosin B (0.25 g/Kg)-   Group 3: mice were injected i.p. with BSA plus BGO 136 (800 mM) in    100 ml PBS, emulsified in (CFA) or erythrosin B (0.5 g/Kg)-   Group 4: mice were treated with BSA in presence of the inhibitor    emulsified in (CFA). These mice received various i.p. doses of BGO    136 400 mM in 0.1 ml of normal PBS or erythrosin B 0.25 g/Kg, 2    doses/week are given as a maintenance dose. (group named    DM=maintenance dose)

These initial immunizations (i.p.) were followed by two subsequentimmunizations with emulsions of BSA alone or in presence of BGO136 400mM or 800 mM, in incomplete Freund's adjuvant. Five days after eachimmunization, blood was collected from each animal, sera was analysed byan ELISA assay.

Detection of Antibodies

Flat-bottomed microtiter plate were coated with BSA (5 ug/ml) inNaHCO₃/Na₂CO₃ 50 mM (pH 9.6) at 37° C. for 2 h. Plates were washed threetimes with (PBS buffer, pH 7.4, 0.5 ml/L Tween-20) and then blocked with400 ul of milk per well overnight, at 4° C. Diluted sera specimens wereadded (100 ul/well) and incubated for 2 h at 37° C. After thoroughwashing, wells were incubated with 100 ul of goat-anti-mouse IgGconjugated with peroxydase for 2 h at 37° C. followed by several washes.TMB substrate solution (100 ul/well), (42 mM TMB in DMSO, 0.1 M citricacid, Na₂HPO₄ 0.2 M and H₂O₂ 30%) was added. Color development wasallowed to proceed for approximately 15 to 30 min, at room temperature.Reaction was terminated by adding 30 ul of 4N H2SO₄. Optical density wasread at 450 nm.

Cell Growth and Proliferation of Jurkat Cell Lines

T cell Jurkat is an acute T cell leukemia obtained from the peripheralblood of human. It was purchassed from ATCC and was grown and maintainedin RPMI™. 1640 medium containing 10% FBS, 2 mM L-glutamine adjusted tocontain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0mM sodium pyruvate. To evaluate the effect of the different inhibitorsof NTPDases on the growth of Jurkat cells, BGO 136, erythrosin, 8 BusATP, AMP and β-γ-Me ATP were added in the RPMI™ 1640 medium in presenceof cells at day=0. Cells counts and viability of cells were determinedon day=5.

Statistical Analysis

Mice were used as test animals. Studies were also performed on thecultures of peripheral blood lymphocytes of donors and on experimentaltumor cell lines. Test results were statistically processed. A one wayvariance analysis was followed; the Statpack® software analysis programwas utilized.

-   *p<0.05-   ** p<0.01-   *** p<0.001

EXAMPLE 1 Presence of NTPDase in Various Normal and Neoplastic LymphoidCells and Inhibitory Activity of NaN3

The presence and activity of NTPDase in PBL cells and in various tumorcells were compared using ATP and ADP as substrates.

As may be seen in FIG. 1, the NTPase signal is more pronounced inneoplastic cells as compared to that in normal lymphocytes (peripheralblood lymphocytes (PBL)).

The levels of the ecto-ATPase and ecto-ADPase functions of NTPDase arepresented in Table 1 below. In agreement with the Western-blots shown inFIG. 1, the levels of NTPDase activity in normal PBL cells is relativelylow as compared to that in tested tumor cell lines. It is noteworthythat in most cases, 10 mM Na azide (NaN3) (a NTPDase inhibitor) causesmore than 50% inhibition of enzyme activity. TABLE 1 NTPDase activity ofvarious intact human normal and tumoral lymphoid cells (nmolesPi/min/10⁶ cells) ATP ATP + NaN3 ADP ADP + NaN3 n = 3 Ramos  20 ± 0.3*** 10 ± 0.0 14.5 ± 2.4***   8 ± 0 Raji  39 ± 6.0***  14 ± 0.0   21 ±0.8***  14 ± 0 JurKat  25 ± 1.5***   7 ± 1.0  9.5 ± 0.8*** 3.5 ± 0.8Mono Mac-1  23 ± 1.0***   7 ± 1.2  7.7 ± 0.2*** 3.8 ± 1.8 U937  29 ±1.0***   8 ± 0.0   11 ± 0.5*** 3.3 ± 0.8 n = 4 PBL 1.0 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.2 ± 0.1n = number of experiments (each in triplicate)***indicates that the difference of NTPDase activity between tumor celllines group and the control PBL group is significant.

EXAMPLE 2 Effect of BGO 136 on NTPDase Activity of PBL

As shown in FIG. 2, with PBL, BGO 136 caused a dose-dependent inhibitionof ATPase and ADPase activities. A significant inhibition (40%) wasobtained with 5 mM BGO 136 whereas with 10 mM, it increased up to 50% ormore. The results are expressed as percent of controls consideringuntreated control samples as 100.

The influence of BGO 136 on human lymphocyte proliferation stimulated byConA™ and LPS™ is shown in FIG. 3. ConA™ is a mitogen specific tolymphocytes T while LPS™ is a mitogen specific to lymphocytes B. BGO 136did not affect the proliferation of non-activated lymphocytes asmeasured by 3H-Thymidine incorporation in DNA. In contrast,proliferation induced by ConA™ was inhibited in a dose-dependent manner,reaching about 10% of the positive control values with 10 mM BGO 136.The BGO 136 inhibition of LPS™-induced proliferation was much lesspronounced. One has to take into account that cell-induced proliferationas expected is relatively low.

In PBL proliferation assays presented in the Examples below, ConA™ onlywas used to specifically stimulate T lymphocyte proliferation.

A positive correlation (0.89 for ATPase and 0.96 for ADPase) betweencell proliferation presented in FIG. 3 and NTPDase activity presented inFIG. 2 was established in the presence of the three concentrations ofBGO 136 is illustrated in FIG. 4.

EXAMPLE 3 Effect of BGO 136 on Humoral Response

As shown in FIG. 5, the levels of albumin antibodies were reduced asmuch as 50% by a single dose BGO 136 (800 mM). Repeated injections ofBGO 136 appeared even more efficient (group DM in FIG. 5).

The experiments were repeated in several series of mice and producedessentially the same pattern of inhibition. The results from onerepresentative experiment are presented in FIG. 5.

No toxic or allergic reactions were observed during the BGO 136administration (two months).

EXAMPLE 4 Effect of Erythrosin B on PBL NTPDase Activity and PBLProliferation

The effect of erythrosin B, a NTPDase inhibitor not structurally relatedto BGO 136, on lymphocyte T activity was also observed to determine itsability to influence immune response of T-cells. Results presented inFIGS. 6-7-8 show a dose-dependent inhibition of lymphocyte proliferation(FIG. 7) and NTPDase activity (FIG. 6). A high correlation wasestablished between these two parameters (FIG. 8).

EXAMPLE 5 Effect of Erythrosin B on Humoral Response

As shown in FIG. 9, a significant decrease in the levels of antibodiesto albumin following administration of erythrosin B can be observedespecially with multiple injections to sustain the concentration of theinhibitor in the animal (group DM in FIG. 9).

EXAMPLE 6 Effect of 8 BUS-ATP on PBL NTPDase Activity and PBLProliferation

A pronounced dose-dependent inhibition of the NTPDase activity by 8BUS-AMP was observed (FIG. 10). 8 BUS-ATP reduced cell proliferationinduced by ConA™ (FIG. 11). A very good correlation could be establishedbetween these two parameters (FIG. 12).

EXAMPLE 7 Effect of 8 BUS-AMP on PBL NTPDase Activity and PBLProliferation

A pronounced dose-dependent inhibition of the NTPDase activity by 8BUS-AMP was observed (FIG. 13). 8 BUS-ATP reduced cell proliferationinduced by ConA™ (FIG. 14). A high correlation was established betweenthese two parameters (FIG. 15).

EXAMPLE 8 Effect of β-γ-Me ATP on PBL NTPDase Activity and PBLProliferation

As shown in FIG. 16, NTPDase was inhibited in a dose-dependent manner.β-γ-Me ATP reduced cell proliferation induced by ConA™ (FIG. 17). Again,a high correlation was established between these two parameters (FIG.18).

EXAMPLE 9 Effect of BGO 136, Erythrosin, 8 BUS ATP, AMP and β-γ-Me ATPon Jurkat Cells' Proliferation

FIG. 19 shows the influence of the different inhibitors on neoplastic Tlymphocytes, namely Jurkat cells which are derived from human leukemiacells. BGO 136, erythrosin, 8 BUS ATP and AMP exerted a marked reductionof cell proliferation. A less pronounced but significant effect wasobserved with β-γ-Me ATP.

EXAMPLE 10 Comparison of Effect of NTPDase Inhibitors with Compound ofthe Prior Art on Graft vs. Host Reaction

Table 2 below shows the efficiency of NTPDase inhibitors in preventingmice spleen growth following cell transplant according to the Bundickmodel. Mice were injected their parents' spleen cells. 21 days later,their spleen was weighed. Activation of Th2 (T helper cells) cells. Thetreatment groups are cyclosporin, BGO, and AMP. Bold figures in Table 2correspond to results originally obtained by Bundick. The “No treatmentgroup” is constituted of normal mice. The “positive” group isconstituted of mice having undergone spleen tissue transplantation andhaving been injected with the carrier only (PBS™) without inhibitor. Theother groups are constituted of mice having undergone spleen tissuetransplantation and having been injected with the BGO 136, AMP or thecyclosporine, respectively. The spleen of all mice was then removed andweighed. The weigh of treated mice was then compared with that of the“No treatment” mice. Transplanted spleens of mice treated withcyclosporine and BGO 136 are significantly smaller than those of thepositive control mice (ANOVA<5).

As may be seen in this table, BGO 136 was more efficient thancyclosporine in preventing spleen growth following transplantation.TABLE 2 INFLUENCE OF VARIOUS INHIBITORS ON SPLEEN WEIGH AFTERTRANSPLANTATION splenomegaly Groups Mice mg/g of b. weight suppression %No treatment 6 2.1 + 0.23 100 2.7 + 0.35 Positive 8 7.1 + 1.11 07.7 + 1.65 BGO 8 4.3 + 0.29 56 AMP 8 6.7 + 0.82 8 Cyclosporine 6 5.1 +0.09 40

CONCLUSION

The present invention showed a close correlation between inhibition ofNTPDase activity and inhibition of immune responses of normal immunecells and neoplastic immune cells. All tested NTPDase inhibitors (i.e.BGO 136, erythrosin B, 8 Bus ATP, 8 Bus AMP and β-γ-Me ATP) decrease theNTPDase activity, and lymphocytic activity including proliferation andhumoral response in normal T lymphocyte.

All the NTPDase inhibitors tested herein were shown to inhibit thegrowth of leukemic T lymphocyte cells (Jurkat cells) in culture.

As was seen in Table 1, the NTPDase activity in neoplastic lymphoidcells is 10 times higher than that in normal lymphocytes (exemplifiedherein by PBL). NTPDase is therefore inducible in tumoral lymphoidcells. It is reasonably predictable therefore that since NTPDaseinhibitors were able to inhibit humoral response in PBL cells, it will afortiari inhibit this response in tumoral lymphoid cells.

Mice were shown to be a reliable and predictive model for human in theuse of immunosuppressive agents in graft transplant (i.e. cyclosporine).The graft transplant rejection mechanism therefore appears to be similarin mice and human. Furthermore, with regards particularly to NTPDase, aswas shown in Benrezzak et al. 2000, human, mice (and pigs) have verysimilar biochemical profiles with regards to NTPDase activity andlocalization. Results presented herein obtained with mice may reasonablypredict that similar results will be obtained in human for grafttransplant and inhibition of immune cell activity involving NTPDaseactivity.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

REFERENCES

-   [1] Abbrachio, M. P. and Burnstock, G. (1998) Jpn. J. Pharmacol. 78,    113-145.-   [2] Williams, M. and Jarvis, M. J. (2000) Biochem. Pharmacol. 59,    1173-1185.-   [3] Drury, A. N. and Szent-Györgyi, A. (1929) J. Physiol. Lond. 68,    23-237.-   [4] Bennet, D. W. and Drury, A. N. (1931) J. Physiol. Lond.    72,288-320.-   [5] Holton, F. A. and Holton, P. (1954) J. Physiol. Lond.    126,124-140.-   [6] Holton, P. (1959) J. Physiol. Lond. 145, 494-504.-   [7] Burnstock, G. (1969) Pharmacol. Rev. 21, 247-324.-   [8] Su, C.; Bevan, J. A. and Burnstock, G. (1971) Science 173,    336-338.-   [9] Langer, S. Z. and Pinto, E. B. (1976) J. Pharmacol. Exp. Ther.    196, 697-713.-   [10] Burnstock, G. (1976) J. Theor. Biol. 62, 491-503.-   [11] Burnstock, G. (1971) Nature 229, 282-283.-   [12] Burnstock, G. (1978) In: Cell membrane receptors for drugs and    hormones: A multidisciplinary approach, (Straub, R. W. and Bolis,    L.; Eds.), Raven Press, New York, pp. 108-118.-   [13] Coade, S. B. and Pearson, J. D. (1989) Circ. Res. 65, 531-537.-   [14] Beaudoin, a. r.; grondin, G.; enjyoji, K.; robson, S. C.;    sévigny, J.; fischer, B. and gendron, F. P. (2000) in: Ecto-ATPases    and related ectonucleotidases. (Vanduffel, L. and Lemmens, R.; Eds),    Shaker Publishing B. V.; The Netherlands, pp. 125-135.-   [15] Burnstock, G. (1972) Pharmacol. Rev. 24, 510-569.-   [16] Lloyd, h. g. e. and stone, T. W. (1983) Proc. Physiol. Soc.    57p.-   [17] White, T. D. (1988) Pharmacol. Ther. 38, 129-168.-   [18] Williams, M. (1990) Ann. N.Y. Acad. Sci. 603, 93-107.-   [19] Sneddon, P. and Burnstock, G. (1984) Eur. J. Pharmacol. 100,    85-90.-   [20] Sneddon, P. and Westfall, D. P. (1984) J. Physiol. Lond. 347,    561-580.-   [21] Burnstock, G. (1986) Prog. Brain Res. 68,193-203.-   [22] Kirkpatrick, K. and burnstock, G. (1987) Eur. J. Pharmacol.    138, 207-214.-   [23] Vizi, E. S. and Burnstock, G. (1988) Eur. J. Pharmacol. 158,    69-77.-   [24] Westfall, D. P.; Sedaa, K. O.; Shinozuka, K.; Bjun, R. A. and    Buxton, I. L. (1990) Ann. N.Y. Acad. Sci. 603, 300-310.-   [25] Von Kugelgen, I. and Starke, K. (1994) Naunyn-Schmiedebergs    Arch. Pharmacol. 350,123-129.-   [26] Kennedy, C.; McLaren, G. J.; Westfall, T. D. and    Sneddon, P. (1996) Ciba Foundation Symposium 198,223-235.-   [27] Brock, J. A.; Bridgewater, M. and Cunnane, T. C. (1997) Br. J.    Pharmacol. 120, 769-776.-   [28] Richardson, P. J. and Brown, S. J. (1987) J. Neurochem. 48,    622-630.-   [29] Ding, Y.; Cesare, P.; Drew, L.; Nikitaki, D. and    Wood, J. N. (2000) J. Auton. Nerv. Syst. 81, 289-294.-   [30] Hamilton, S. G. and McMahon, S. B. (2000) J. Auton. Nerv. Syst.    81, 187-194.-   [31] Hamilton, S. G.; Wade, A. and McMahon, S. B. (1999) Br. J.    Pharmacol. 126, 326-332.-   [32] Unswoth, c. d. and johnson, R. G. (1990) Ann. N.Y. Acad. Sci.    603, 353-363.-   [33] Uvnas, B. (1974) Life Sci. 14, 2355-2366.-   [34] Roman, R. M.; wang, Y.; lidofsky, S. D.; feranchak, A. P.;    Iomri, N.; scharschmidt, B. F. and fitz, J. G. (1997) J. Biol. Chem.    272, 21970-21976.-   [35] Sprague, r. s.; ellsworth, M. L.; stephenson, A. H.;    kleinhenz, M. E. and lonigro, A. J. (1998) Am. J. Physiol. 44,    H1726-H1732.-   [36] Sugita, m.; yue, Y. and foskett, J. F. (1998) EMBO J. 17,    898-908.-   [37] Beaudoin, a. r.; grondin, G. and gendron, F. P. (1999) Prog.    Brain Res. 120, 387-395.-   [38] Taylor, a. l.; kudlow, B. A.; maus, K. L.; gruenert, D. C.;    guggino, W. B. and schwiebert, E. M. (1998) Am. J. Pharmacol. 275,    C1391-C1406.-   [39] Wilson, p. d.; hovater, J. S.; casey, C. C.; fortenberry, J. A.    and schwiebert, E. M. (1999) J. Am. Soc. Nephro. 10, 219-229.-   [40] Schlosser, S. F.; Burgstahler, A. D. and    Nathanson, M. H. (1996) Proc. Natl. Acad. Sci. USA 93, 9948-9953.-   [41] Lidofsky, S. (1997) Hepatology 25, 778-779.-   [42] Queiroz, g.; gebicke-haerter, P. J.; schobert, A.; starke, K.    and Von kugelgen, L. (1997) Neurosci. 78, 1203-1208.-   [43] Guthrie, p. b.; knappenberger, J.; segal, M.; bennett, M. V.;    charles, A. C. and kater, S. B. (1999) J. Neurosci. 19, 520-528.-   [44] Rojas, e.; pollard, H. B. and heldman, E. (1985) FEBS Letters    185, 323-327.-   [45] Xu, y. p.; duarte, E. P. and forsberg, E. J. (1991) J.    Neurochem. 56, 1889-1896.-   [46] Weil-malherbe, h. and bone, A. D. (1958) Biochem. J. 70, 14-22.-   [47] Marquardt, d. l.; gruber, H. E. and wasserman, S. I. (1994)    Proc. Natl. Acad. Sci. USA 81, 6192-6196.-   [48] Rapaport, e. and fontaine, J. (1989) Biochem. Pharmacol. 38,    4261-4266.-   [49] Sperlagh, b.; hasko, G.; nemeth, Z. and vizi, E. S. (1998)    Neurochem. Int. 33, 209-215.-   [50] Fredholm, B. B.; Abbracchio, M. P.; G. Burnstock, G.; Daly, J.    W.; Harden, T. K.; Jacobson, K. A.; Leff, P. and Williams, M. (1994)    Pharmacol. Rev. 46,143-156.-   [51] Tucker, A. L. and Linden, J. (1993) Cardiovasc. Res. 27, 62-67.-   [52] Dubyak, G. R. and El-Moatassim, C. (1993) Am. J. Physiolgy 265,    C577-C606.-   [53] Stoop, R.; Thomas, S.; Rassedren, F.; Kawashima, E.; Buell, G.;    Surprenant, A. and North, R. (1999) Mol. Pharmacol. 56, 973-981.-   [54] Khakh, B. S.; Burnstock, G.; Kennedy, C.; King, B. F.;    North, R. A.; Seguela, P.; Voigt, M. and Humphrey, P. P. A. (2001)    Pharmacol. Rev. 53(1), 107-118.-   [55] Weisman, G. A.; Turner, J. T.; Clarke, L. L.; Gonzalez, F. A,    Otero, M.; Garrad, R. C. and Erb, L. (1997) in: Ecto-ATPases,    (Plesner, L.; Kirley, T. L. and Knowles, A. F.; Eds.), Plenum Press.    New York, USA, pp. 231-237.-   [56] Evans, R. J.; Lewis, C.; Virgilio, C.; Lundstrom, K.; Buell,    G.; Surprenant, A. and North, R. A. (1996) J. Physiol. Lond. 497,    413-422.-   [57] Vulchanova, L.; arvidsson, U.; riedl, M.; wang, J.; buell, G.;    surprenant, A.; north, R. A. and elde, R. (1996) Proc. Natl. Acad.    Sci. USA 93, 8063-8067.-   [58] Boarder, m. r. and hourani, s. m. o. (1998) Trends Pharmacol.    Sci. 19, 99-107.-   [59] Nicke, A.; Baumert, H. G.; Rettinger, J.; Eichele, A.;    Lambrecht, G.; Mutschler, E. and Schmalzing, G. (1998) EMBO J. 17,    3016-3028.-   [60] Fedan, J. S.; Dagirmanjian, J. P.; Attfield, M. A. and    Chideskel, E. W. (1990) J. Pharmacol. Exp. Ther. 255, 46-51.-   [61] Nakazawa, K.; Fujimori, K.; Takanakna, A. and    Inoue, K. (1990) J. Physiol. Lond. 428, 257-272.-   [62] Sela, D.; Ram, E. and Atlas, D. (1991) J. Biol. Chem. 266,    17990-17994.-   [63] Berridge, M. J. and Irvine, R. F. (1989) Nature 341, 197-205.-   [64] Gordon, J. L. (1986) Biochem. J. 233, 309-319.-   [65] Surprenant, A.; Rassendren, F.; Kawashima, E.; North, R. A. and    Buell, G. (1996) Science 272, 735-738.-   [66] Ferrari, D.; Chiozzi, P.; Falzoni, S.; Dal Susino M.;    Melchiorri, L.; Baricordi, O. R. and Di Virgilio, F. (1997) J.    Immunol. 15, 1451-1458.-   [67] Solle, M.; Labasi, J.; Perregaux, D. G.; Stam, E.; Petrushova,    N.; Koller, B. H.; Griffiths, R. J. and Gabel, C. A. (2001) J. Biol.    Chem. 276(1), 125-132.-   [68] Sneddon, P.; Westfall, T. D.; Todorov, L. D.;    Mihaylova-Todorova, S.; Westfall, D. P. and Kennedy, C. (1999) Prog.    Brain Res. 120, 11-20.-   [69] Kennedy, C. and Leff, p. (1995) Trends Pharmacol. Sci. 16,    168-174.-   [70] North, R. A. (1996) Sem. Neurosci. 8,187-194.-   [71] Torres, G.; Egan, T. and Voigt, M. (1999) J. Biol. Chem. 274,    6653-6659.-   [72] Lewis, C.; Neidhart, S.; Holy, C.; North, R. A.; Buell, G. and    Surprenant, A. (1995) Nature (Lond.) 377, 432-435.-   [73] Cook, S. P.; Vulchanova, L.; Hargreaves, K. M.; Elde, R. and    McCleskey, E. W. (1997) Nature (Lond.) 387, 505-508.-   [74] Radford, K. M.; Virginio, C.; Surprenant, A.; North, R. A. and    Kawashima, E. (1997) J. Neurosci. 17, 6529-6533.-   [75] Vulchanova, L.; Riedi, M. S.; Shuster, S. J.; Buell, G.;    Surprenant, A.; North, A. R. and Elde, R. (1997) Neuropharmacology    36, 1229-1242.-   [76] Haines, W. R.; Torres, G. E.; Voigt M. M. and    Egan, T. M. (1999) Mol. Pharmacol. 56, 720-727.-   [77] Le, K. T.; Boue-Grabot, E.; Archambault V. and    Séguéla, P. (1999) J. Biol. Chem. 274, 15415-15419.-   [78] Torres, G. E.; Egan, T. M. and Voigt, M. M. (1998) Biochemistry    37, 4845-4851.-   [79] Le, K. T.; Babinski, K. and Séguela, P. (1998) J. Neurosci. 18,    7152-7159.-   [80] Hourani, S. M. O. and Hall, D. A. (1994) Trends Pharmacol. Sci.    15, 103-108.-   [81] Communi, D.; Govaerts, C.; Parmentier, M. and    Boeynaems, J. M. (1997) J. Biol. Chem. 272, 31969-31973.-   [82] Leon, C.; Hechler, B.; Vial, C.; Leray, C.; Cazenave, J.-P. and    Gachet, T. C. (1997) FEBS Lett. 403, 26-30.-   [83] Nicholas, R. A.; Watt, W. C.; Lazarowski, E. R.; Li, Q. and    Harden, T. K. (1996) Mol. Pharmacol. 50, 224-229.-   [84] Boarder, M. R.; Weisman, G. A.; Turner, J. T. and    Wilkinson, G. F. (1995) Trends Pharmacol. Sci. 16,133-138.-   [85] Harden, T. K.; Boyer, J. L. and Nicholas, R. A. (1995) Ann.    Rev. Pharmacol. Thoxicol. 35, 541-579.-   [86] Yang, C. M.; Tsai, Y.-J.; Pan, S.-L.; Tsai, C.-T.; Wu, W.-b.;    Chiu, C.-T.; Luo, S.-F. and Ou, J. T. (1997) Naunyn-Schmiedeberg's    Arch. Pharmacol. 356, 1-7.-   [87] Erb, L.; Lustig, K. D.; Sullivan, D. M.; Turner, J. T. and    Weisman, G. A. (1994) Proc. Natl. Acad. Sci. USA 90, 10449-10453.-   [88] Boyer, J. L.; Delaney, S. M.; Villanueva, D. and Harden, T. K.    (2000). Mol. Pharmacol. 57, 805-810-   [89] Hollopeter, G.; Jantzen, H. M.; Vincent, D.; Li, G.; England,    L.; Ramakrishnan, V.; Yang, R. B.; Nurden, P.; Nurden, A.;    Julius, D. and Conley, P. B. (2000) Nature 409, 202-207.-   [90] Tu, M.-T.; Luo, S.-F.; Wang, C.-C.; Chien, C.-S.; Chiu, C.-T.;    Lin, C.-C. and Yang, C. M. (2000) Br. J. Pharmacol. 129, 1481-1489.-   [91] Erb, L.; Liu, J.; Ockerhausen, J.; Kong, Q.; Garrad, R. C.;    Griffin, K.; Neal, C.; Krugh, B.; Santiago-Pérez, L. I.;    Gonzalez, F. A.; Gresham, H. D.; Turner, J. T. and    Weisman, G. A. (2001) J. Cell. Biol. 153, 491-501.-   [92] Short, S. M.; Boyer, J. L. and Juliano, R. L. (2000) J. Biol.    Chem. 275, 12970-12977.-   [93] Zimmermann, H. (2000) Naunyn-Schmiedeberg's Arch. Pharmacol.    362, 299-309.-   [94] Enjyoji, K.; Sévigny, J.; Lin, Y.; Fenette, P. S.; Christie, P.    D.; Schulte am Esch II, J.; Imai, M.; Edelberg, J. M.; Rayburn, H.;    Lech, M.; Beller, D. L.; Csizmadia, E.; Wagner, D. D.; Robson, S. C.    and Rosenburg, R. D. (1999) Nature Med. 5,101-1017.-   [95] LeBel, D.; Poirier, G. G.; Phaneuf, S.; St-Jean, P.; Laliberte,    J.-F. and Beaudoin, A. R. (1980) J. Biol. Chem. 255, 1227-1233.-   [96] Zimmermann, H. (1992) Biochem. J. 285, 345-365.-   [97] Ziganshin, A. U.; Hoyle, C. H. V and Burnstock, G. (1994) Drug    Dev. Res. 32,134-146.-   [98] Plesner, L. (1995) Int. Rev. Cytol. 158,141-214.-   [99] Beaudoin, A. R.; Sévigny, J. and Picher, M. (1996) In:    Biomembranes Vol. 5. (Lee, A. G.; Ed.). JAI Press Inc. Greenwich,    pp. 367-399.-   [100] Vlajkovic, S. M.; Thome, P. R.; Hously, G. D.; Munoz, D. J. B.    and Kendrick, I. S. (1998) Neuroreport 9, 1559-1565.-   [101] Culic, O.; Sabolic, I. and Zanic-Grubisic, T. (1990) Biochim.    Biophys. Acta 1030, 143-151.-   [102] Meyerhof, 0. (1945) J. Biol. Chem. 157, 105-119.-   [103] Kalckar, H. M. (1944). J. Biol. Chem. 153, 355-367.-   [104] Ribeiro, J. M. C. (1987) Ann. Rev. Entomol. 32, 463-478.-   [105] Ribeiro, J. M. C. (2000) Med. Vet. Entom. 14,142-148.-   [106] Vasconcelos, E. G.; Nascimento, P. S.; Meirelles, M. N. L.;    Verjovski-Almeida, S. and Ferreira, S. T. (1993) Mol. Biochem.    Parasitol. 58, 205-214-   [107] Anich, M.; Fanta, N.; Mancilla, M.; Kettlun, A, M,    Velenzuela, M. A. and Traverso-Cori, A. (1990) Phytochemistry 29,    1411-1415.-   [108] Thomas, C.; Sun, Y.; Naus, K.; Lloyd, A. and Roux, S. (1999)    Plant Physiol. 119, 543-551.-   [109] Bhargava, T.; Datta, S.; Ramachandran, V.; Ramakrishnan, R.;    Roy, R. K.; Sankaran, K. and Subrahmanyam, Y. V. B. K. (1995) Curr.    Sci. India 68, 293-300.-   [110] Curdova, E.; Jechova, V. and Hostalek, Z. (1982) Folia    Microbiol. 27, 159-166.-   [111] Trilisenko, L. V.; Novotna, J.; Erban, V.; Behal, V. and    Hostalek, Z. (1987) Folia Microbiol. 32, 402-410.-   [112] Vasconcelos, E. G.; Ferraira, S. T.; De Carvalho, T. M. U.; De    Souza, W.; Kettlun, A. M.; Mancilla, M.; Valenzuela, M. A. and    Verjovski-Almeida, S. (1996) J. Biol. Chem. 271, 22139-22145.-   [113] Berlutti, F.; Casalino, M.; Zagaglia, C.; Fradiana, P. A.;    Visca, P. and Nicoletti, M. (1998) Inf. Immun. 66, 4957-4964.-   [114] Yagi, K.; Arai, Y.; Kato, N.; Hirota, K. and Miura, Y. (1989)    Eur. J. Biochem. 180, 509-513.-   [115] Côté, Y.-P.; Picher, M.; St-Jean, P.; Béliveau, R.; Potier, M.    and Beaudoin, A. R. 1991. Biochim. Biophys. Acta 1078, 187-191.-   [116] Beaudoin, A. R.; Sévigny, J.; Grondin, G.; Daoud, S. and    Levesque, F. P. (1997) Am. J. Physiol. 273: H673-H681.-   [117] Sévigny, J.; Levesque, F. P.; Grondin, G. and Beaudoin, A. R.    (1997). Biochim. Biophys. Acta 1334, 73-88-   [118] Picher, M.; Côté, Y. P.; Béliveau, R.; Potier, M. and    Beaudoin, A. R. (1993) J. Biol. Chem. 268, 4699-4703.-   [119] Sévigny, J.; Picher, M.; grondin, G. and    Beaudoin, A. R. (1997) Am. J. Physiol. 272, L939-L950.-   [120] Sévigny, J.; grondin, G.; Gendron, F. P.; roy, J. and    Beaudoin, A. R. (1998) Am. J. Physiol. 275, G473-G482.-   [121] Leclerc, M.-C.; Grondin, G.; Gendron, F. P.; Sévigny, J. and    Beaudoin, A. R. (2000) Arch. Biochem. Biophys. 377, 373-378.-   [122] Sévigny, J.; Robson, S. C.; Waelkens, E.; Csizmadia, E.;    Smith, R. N. and Lemmens, R. (2000) J. Biol. Chem. 275, 5640-5647.-   [123] Zimmermann, H. and Braun, N. (1996) J. Auton. Pharmacol. 16,    397-400.-   [124] Wang, T. F. and Guidotti, G. (1998) Brain Res. 790, 318-322.-   [125] Maliszewski, C. R.; Delespesse, G. J. T.; Schoenborn, M. A.;    Armitage, R. J.; Fanslow, W. C.; Nakajima, T.; Baker, E.;    Sutherland, G. R.; Poindexter, K.; Birks, C.; Alpert, A.; Friend,    D.; Gimpel, S. D. and Gayle III, R. B. (1994) J. Immunol. 153,    3574-3583.-   [126] Benrezzak, O.; Grondin, G.; Sévigny, J.; Gendron, F. P.;    Rousseau. E.; D'Orléans-Juste, P. and Beaudoin, A. R. (1999) Arch.    Biochem. Biophys. 370, 314-322.-   [127] Valenzuela, M. A.; Lopez, J.; Depix, M.; Mancilla, M.;    Kettlun, A. M.; Catalan, L.; Chiong, M.; Garrido, J. and    Traverso-Cori, A. (1989) Comp. Biochem. Physiol. 93B, 509-513.-   [128] Papamarcaki, T. and Tsolas, O. (1990) Mol. Cell. Biochem. 97,    1-8.-   [129] Magocsi, M. and Penniston, J. T. (1991) Biochim. Biophys. Acta    1070, 163-172.-   [130] Pieber, M.; Valenzuela, M. A.; Kettlun, A. M.; Mancilla, M.;    Aranda, E.; Collados, L. and Traverso-Cori, A. (1991) Comp. Biochem.    Biophys. 100B, 281-285.-   [131] Lemmens, R.; Kupers, L.; Sévigny, J.; Beaudoin, A. R.;    Grondin, G.; Kittel, A.; Waelkens, E. and Vanduffel, L. (2000)    Am. J. Physiol. 278, F978-F988.-   [132] Handa, M. and Guidotti, G. (1996) Biochem. Biophys. Res. Comm.    218, 916-923.-   [133] Schulte, J. A. E, Sévigny, J.; Kaczmarek, E.; Siegel, J. B.;    Imai, M.; Koziak, K.; Beaudoin, A. R. and Robson, S. C. (1999)    Biochemistry 38, 2248-2258.-   [134] Smith, T. M.; Carl, S. A. L. and Kirley, T. L. (1999)    Biochemistry 38, 5849-5857.-   [135] Drosopoulos, J. H. F.; Broekman, M. J.; Islam, N.;    Maliszewski, C. R.; Gayle, R. B. and Marcus, A. J. (2000)    Biochemistry 39, 6936-6943.-   [136] Grinthal, A. and Guidotti, G. (2000) Biochemistry 39, 9-16.-   [137] Asai, T.; Miura, S.; Sibley, L. D.; Okabayashi, H. and    Takeuchi, T. (1995) J. Biol. Chem. 270, 11391-11397.-   [138] Kegel, B.; Braun, N.; Heine, P.; Maliszewski, C. R. and    Zimmermann, H. (1997) Neuropharmacol. 36, 1189-1200.-   [139] Stout, J. G. and Kirley, T. L. (1996) Biochemistry 35,    8289-8298.-   [140] Wang, T. F.; Ou, Y. and Guidotti, G. (1998) J. Biol. Chem.    273, 24814-24821.-   [141] Wang, T. F.; Rosenberg, P. A. and Guidotti, G. (1997) Brain    Res. Mol. Brain Res. 47, 395-302.-   [142] Sévigny, J.; Dumas, F. and Beaudoin, A. R. (1997) In:    Ecto-ATPase. Recent progress on structure and function. (Plesner,    L.; Kirley, T. L. and Knowles, A. F.; Eds.) Plenum Press. New York,    294 P.-   [143] Chadwick, B. P. and Frischauf, A.-M. (1998) Genomics 50,    357-367.-   [144] Schoenborn, M. A.; Jenkins, N. A.; Copeland, N. G.;    Gilbert, P. J.; Gayle III, R. B. and Maliszewski C. R. (1998)    Cytogenet. Cell Genet. 81, 287-289.-   [145] Zimmermann, H.; Beaudoin, A. R.; Bollen, M.; Goding, J. W.;    Guidotti, G.; Kirley, T. L.; Robson, S. C. and Sano, K. (2000). In:    Ecto-ATPases and related ectonucleotidases. (Vanduffel, L. and    Lemmens, R.; Eds) Shaker Publishing B. V.; The Netherlands. Pp 1-8.-   [146] Côté, Y. P.; Filep, J. G.; Battistini, B.; Gauvreau, J.;    Sirois, P. and Beaudoin, A. R. (1992) Biochim. Biophys. Acta    1139,133-42.-   [147] Kirley, T. L. (1997) J. Biol. Chem. 272, 1076-1081.-   [148] Marcus, A. J.; Broekman, M. J.; Drosopoulos, J. H. F.; Islam,    N.; Alyonycheva, T. N.; Safier, J. B.; Hajjar, K. A.; Posnett, D.    N.; Schoenborn, M. A.; Schooley, K. A.; Gayle III, R. B. and    Maliszewski, C. R. (1997) J. Clin. Invest. 99, 1351-1360.-   [149] Koziak, K.; Sévigny, J.; Robson, S. C.; Siegel, J. B. and    Kaczmarek, E. (1999) Thromb. Haemo. 82, 1538-1544.-   [150] Kansas, G. S.; Wood, G. S. and Tedder, T. F. (1991) J.    Immunol. 146, 2235-2244.-   [151] Frassetto, S. S.; Dias, R. D. and Sarkis, J. J. F. (1993) Mol.    Cell. Biochem. 129, 47-55.-   [152] Marcus, A. J. (1996) In: Disorders of Hemostasis, chap. 4    (O. D. Ratnoff and C. D. Forbes, ed.), pp. 79-137, Philadelphia,    U.S.A.-   [153] Hirota, K.; Saski, N.; Yagi, K. and Miura, Y. (1987) Thromb.    Res. 45, 201-209.-   [154] Kaczmarek, E.; Koziak, K.; Sévigny, J.; Siegel, J. B.;    Anrather, J.; Beaudoin, A. R.; Bach, F. H. and    Robson, S. C. (1996) J. Biol. Chem. 271, 33116-33122.-   [155] Koyamada, N.; Miyatake, T.; Candinas, D.; Hechenleither, P.;    Siegel, J.; Hancock, W. W.; Bach, F. H. and Robson, S. C. (1996)    Transplantation 62, 1739-1743.-   [156] Imai, M.; Takigami, K.; Guckelberger, O.; Enjyoji, K.; Neal    Smith, R.; Lin, Y.; Csizmadia, E.; Sévigny, J.; Rosenberg, R. D.;    Bach, F. H. and Robson, S. C. (1999) Mol. Med. 5, 743-752.-   [157] Braun, N.; Zhu, Y, Krieglstein, J.; Culmsee, C. and    Zimmermann, H. 1998. J. Neurosci. 18, 4891-4900.-   [158] Rongen, G. A.; Floras, J. S.; Lender, J. W.; Thien, T.;    Smits, P. (1997) Clin. Sci. 92, 13-24.-   [159] Di Virgilio, F.; Bronte, V.; Collavo, D. and    Zanovello, P. (1989) J. Immunol. 143, 1955-1960.-   [160] Filippini, A.; Taffs, R. E.; Agui, T. and    Sitkovsky, M. V. (1990) J. Biol. Chem. 265, 334-340.-   [161] Antonysamy, M. A.; Moticka, E. J. and Ramkumar, V. (1995) J.    Immunol. 155, 2813-2821.-   [162] Krishnaraj, R. (1992) Cell Immunol. 141, 306-322.-   [163] Krishnaraj, R. (1992b) Cell Immunol. 144, 11-21.-   [164] Bajpai, A. and Brahmi, Z. (1993) Cell. Immunol. 148,130-143.-   [165] Dombrowski, K. E.; Cone, J. C.; Bjorndahl, J. M. and    Phillips, C. A. (1995) Cell. Immunol. 160, 199-204.-   [166] Correale, P.; Giuliano, M.; Tagliaferri, P.; Guarrusi, R.;    Caraglia, M.; Marinetti, M. R.; Iezzi, T.; Bianco, A. R. and    Procoplo, A. (1995) Res. Comm. Mol. Pathol. Pharmacol. 87, 67-69.-   [167] Imai, M.; Goepfert, C.; Kaczmareck E. and Robson, S. C. (2000)    Biochem. Biophys. Res. Comm. 270, 272-278.-   [168] Clifford, E. E.;. Martin, K. A.; Dalal, P.; Thomas, R. and    Dubyak, G. R. (1997) Am. J. Physiol. 42, C973-C987.-   [169] Dzhandzhugazyan, K. N.; Kirkin, A. F.; Straten, P. T. and    Zeuthen, J. (1998) FEBS Left. 430, 227-230.-   [170] Gendron, F. P.; Halbfinger, E.; Fischer, B.; Duval, M.;    D'Orléans-Juste, P. and Beaudoin, A. R. (2000a) J. Med. Chem. 43,    2239-2247.-   [171] Gendron, F. P.; Halbfinger, E.; Fischer, B.; and    Beaudoin A. R. (2000) In: Purine and pyrimidine metabolism in man X    (Eds. E. Zoref-Shani and 0. Sperling). Adv. Exp. Med. Biol.    486,119-123.-   [172] Picher, M.; Sévigny, J.; D'Orléans-Juste, P. and    Beaudoin, A. R. (1996) Biochem. Pharmacol. 51, 1453-1460.-   [173] Fischer, B.; Chulkin, A.; Boyer, J. L.; Harden, K. T.;    Gendron, F. P.; Beaudoin, A. R.; Chapal, J.; Hillaire-Buys, D. and    Petit, P. (1999) J. Med. Chem. 42, 3636-3646.-   [174] Halbfinger, E.; Major, D. T.; Ritzman, M.; Ubl, J. J.; Reiser,    G.; Boyer, J. L.; Harden, K. T. and Fischer, B. (1999) J. Med. Chem.    42, 5325-5337.-   [175] Fischer, B.; Kabha, E.; Gendron, F. P. and    Beaudoin, A. R. (2000) Nucleosides, Nucleotides & Nucleic Acid 19,    1033-1054.-   [176] Bültmann, R.; Wittenburg, H.; Pause, B.; Kurz, G.; Nickel, P.    and Starke, K. (1996) Naunyn-Schmiedeberg's Arch. Pharmacol. 354,    498-504.-   [177] Tuluc, F.; Bültmann, R.; Glanzel, M.; Wilhelm Frahm, A. and    Starke, K. (1998) Naunyn-Schmiedeberg's Arch. Pharmacol. 357,    111-120.-   [178] Wittenburg, H.; Bültmann, R.; Pause, B.; Ganter, C.; Kurz, G.    and Starke. K. (1996) Naunyn-Schmiedeberg's Arch. Pharmacol. 354,    491-497.-   [179] Bültmann, R.; Pause, B.; Wittenburg, H.; Kurz, G. and    Starke, K. (1996) Naunyn-Schmiedeberg's Arch. Pharmacol. 354,    481-490.-   [180] Parr, C. E.; Sullivan, D. M.; Paradiso, A. M.; Larowski, E.    R.; Burch, L. H.; Olsen, J. C.; Erb, L.; Weisman, G. A.;    Boucher, R. C. and Turner, J. T. (1994) Proc. Nat. Acad. Sci. USA    91, 3275-3279.-   [181] Vlajkovic,. S. M.; Hously, G. D.; Greenwood, D. and Thorne    PR. (1999) Mol. Brain Res. 73, 85-92.-   [182] Zimmermann, H. (1999) Nat. Med. 5, 987-988.-   [183] Bergfeld, g. r. and forrester, T. (1992) Cardiovas. Res. 26,    40-47.-   [184] Knofler, r.; Weissbach, G. and Kuhlisch, E. (1997) Am. J.    Hematol. 56, 259-265.-   [185] Detwiler, t. c. and feinman, R. D. (1973) Biochemistry 12,    2462-2468.-   [186] Li, b. y. and li, W. H. (1998) Acta Pharmacol. Sinica 19,    383-386.-   [187] Bodin, p. and burnstock, G. (1995) Experientia 51, 256-259.-   [188] Bodin, p.; milner, P.; winter, R. and burnstock, G. (1992)    Proc. R. Soc. Lond. 247,131-135.-   [189] Schini, v. b.; hendrickson, H.; heublein, D. M.;    burnett, J. C. and vanhoutte, P. M. (1989) Eur. J. Pharmacol. 165,    333-334.-   [190] Bodin, p. and burnstock, G. (1996) J. Cardiovasc. Pharmacol.    27, 872-875.-   [191] Sedaa, k. o.; bjur, R. A.; schinozuka, K. and    westfall, D. P. (1990) J. Pharmacol. Exp. Ther. 252, 1060-1067.-   [192] Yang, s.; cheek, D. J.; westfall, D. P. and    buxton, I. L. (1994) Circ. Res. 74, 401-407.-   [193] Vizi, e. s.; sperlagh, B. and burnstock, G. (1992) Neurosci.    50, 455-465.-   [194] Katsuragi, t.; tokunaga, T.; ogawa, S.; soejima, O.; sato, C.    and furukawa, T. (1991) J. Pharmacol. Exp. Ther. 259, 513-518.-   [195] Katsuragi, t.; tamesue, S.; sato, C.; sato, Y. and    furukawa, T. (1996) Naunyn-Schmiedebergs Arch. Pharmacol. 354,    796-799.-   [196] Tamesue, s.; sato, C. and katsuragi, T. (1998)    Naunyn-Schmiedebergs Arc. Pharmacol. 357, 240-244.-   [197] Gendron F. P., Benrezzak, O., Krugh, B. W., Kong, Q.,    Weisman, G. A., and Beaudoin A. R. 2002. Purine signalling and    potential new therapeutic approach: possible outcome of NTPDase    inhibition. Current Drug Targets 3(3): 229-245.-   [198] Nahum, V., Zundorf, G., Reiser, G., Levesque, S. A.,    Beeaudoin, A. R., Fischer, B. 2002.    5′-O-(1-Boranotriphosphate)-Adenosine Derivatives as Novel    P2Y₁-Receptor Agonists. (Accepted J. Med. Chem.)

1. A method of screening for a compound useful in the treatment of adisease or condition characterized by an immune cells disorder, whereinsaid cell expresses NTPDases, said method comprising the steps ofcontacting a candidate compound with ecto-nucleoside triphosphatediphosphohydrolase (NTPDase), wherein the candidate compound is selectedif the activity of said NTPDase is reduced in the presence of thecandidate compound as compared to that in the absence thereof.
 2. Amethod as defined in claim 1, wherein said contacting of said candidatecompound with said NTPDase is performed in an immune cell selected fromthe group consisting in normal T lymphocyte, normal B lymphocyte, normalNK cell, normal macrophage, normal monocyte, Jurkat cell, Raji cell,Ramos cell, MonoMac™ cell, K562 cell and U937 cell.
 3. A method asdefined in claim 2, wherein said immune cell is a T lymphocyte.
 4. Amethod as defined in claim 2, wherein said immune cell is a Blymphocyte.
 5. A method as defined in claim 2, wherein said immune cellis a Jurkat cell.
 6. A method as defined in claim 2, wherein said immunecell is a Raji Cell.
 7. A method as defined in claim 2, wherein saidimmune cell is a Ramos cell.
 8. A method as defined in claim 2, whereinsaid immune cell is a MonoMac™ cell.
 9. A method as defined in claim 2,wherein said immune cell is a K562 cell.
 10. A method as defined inclaim 2, wherein said immune cell is a U937 cell.
 11. A method asdefined in claim 2, wherein said immune cell is a NK cell.
 12. A methodfor inhibiting an immune cell activity in a mammal, comprising targetingimmune cells with an effective amount of a NTPDase inhibitor.
 13. Amethod as recited in claim 12, wherein said cells are normallymphocytes.
 14. The method as recited in claim 13, wherein said normalcells are normal T lymphocytes.
 15. The method as recited in claim 14,wherein said activity is the T lymphocyte proliferation.
 16. The methodas recited in claim 13, wherein said normal lymphocytes are normal Blymphocyte.
 17. The method as recited in claim 12, wherein said activityis the production of antibodies.
 18. A method as recited in claim 12,wherein said cells are neoplastic lymphocytes.
 19. The method as recitedin claim 18, wherein said neoplastic lymphocytes are neoplastic Tlymphocytes.
 20. The method as recited in claim 19, wherein saidneoplastic T lymphocytes are Jurkat cells.
 21. The method as recited inclaim 12, wherein said activity is induced by organ or tissuetransplant.
 22. The method as recited in claim 12, wherein said activityis induced by an allergen.
 23. The method as recited in claim 12,wherein said activity is induced in autoimmune diseases.
 24. A method asrecited in claim 12, wherein said NTPDase inhibitor is selected from thegroup consisting of BGO 136, erythrosin B, nucleotide or nucleotidederivative including AMP, 8 Bus-AMP and 8 Bus-ATP, and analoguesthereof.
 25. The method as recited in claim 12, wherein said inhibitoris BGO136 or a BGO136 analogue.
 26. The method as recited in claim 12,wherein said inhibitor is AMP or an AMP analogue.
 27. The method asrecited in claim 12, wherein said inhibitor is 8 Bus-AMP.
 28. The methodas recited in claim 12, wherein said inhibitor is ATP or an ATPanalogue.
 29. The method as recited in 12, wherein said inhibitor is 8Bus-ATP.
 30. The method as recited in claim 12, wherein said inhibitoris erythrosine B or an erytbrosin B analogue.
 31. A method to prevent orreduce the risk of rejection of transplanted tissue or organ, comprisingadministering to said animal an effective amount of NTPDase inhibitor.32. A method as recited in claim 31, wherein said NTPDase inhibitor isselected from the group consisting of BGO 136, erythrosin B, nucleotideor nucleotide derivative including AMP, 8 Bus-AMP and 8 Bus-ATP, andanalogues thereof.
 33. The method as recited in claim 32, wherein saidinhibitor is BGO136 or a BGO136 analogue.
 34. The method as recited inclaim 32, wherein said inhibitor is AMP or an AMP analogue.
 35. Themethod as recited in claim 32, wherein said inhibitor is 8 Bus-AMP. 36.The method as recited in claim 32, wherein said inhibitor is ATP or anATP analogue.
 37. The method as recited in claim 32, wherein saidinhibitor is 8 Bus-ATP.
 38. The method as recited in claim 32, whereinsaid inhibitor is erythrosine B or an erythrosin B analogue.
 39. Themethod as recited in claim 32, wherein said inhibitor is BGO
 136. 40.Composition for use as an immunosuppressive agent in graft transplantcomprising an effective amount of BGO 136 or BGO 136 analogue in apharmaceutically acceptable carrier.
 41. Use of a BGO 136 or BGO 136analogue in the making of a medicament for use as an immunosuppressiveagent in graft transplant.